Electroporation-mediated transient gene expression in isolated scutella of Hordeum vulgare

Electroporation was used to introduce DNA into excised scutella of immature embryos of Hordeum vulgare L. cvs Golden Promise and Delita. Using the firefly luciferase gene as reporter, parameters were analyzed for high transient gene expression while maintaining tissue viability. Enzymatic wounding was necessary for DNA uptake. The optimized protocol involves use of linearized DNA and addition of 15% (w/v) polyethylene glycol at a field strength of 950 V cm⁻¹ and approximately 56 ms pulse length. A one-day preculture was required for obtaining callus after electroporation. Transient gene expression was further demonstrated using the β-glucuronidase gene. Blue spots were detected at the abaxial scutellar surface, indicating that cells competent for somatic embryogenesis are also amenable to transfection by electroporation.     

Regulation of an embryogenic carrot gene (DC 2.15) and identification of its active promoter sites

According to previous studies the expression of the geneDC 2.15 is induced in cultured carrot cells after a transfer to an auxin-free medium, where somatic embryo development occurs. This embryogenic gene encodes a prolinerich protein, which resembles proteins involved in auxin-controlled developmental processes. To understand the mechanism underlying the regulation ofDC 2.15, an experimental approach has been employed which allows the direct identification of theDC 2.15 promoter structure by applying PCR techniques. We demonstrate the presence of five distinct promoter sequences highly similar in structure, but slightly different in a common region of about 15 nucleotides, which contain the binding site for the GATA factor originally found in the human HOX gene. Activity of each promoter structure was assessed in developing somatic embryos containing the specific sequence fused to the -glucuronidase (GUS) reporter gene. For two of the five promoter structures a drastic increase in activity was registered during the torpedo stage while the remaining three were inactive throughout the stages of somatic embryogenesis.     

Chronic effects of low insecticide concentrations on freshwater caddisfly larvae

The insecticide load in surface waters does not ordinarily reach concentrations acutely toxic to aquatic fauna. The effects of the low insecticide concentrations typical of natural habitats are still not clear, for they often appear only after relatively long exposure times. To test such a situation, the insecticides lindane and parathion were introduced into a static-with-renewal outdoor aquaria system at concentrations about four and five orders of magnitude lower than their respective 96-h LC₅₀s, and their chronic (about 90 days) effects on the survival rate of freshwater caddisfly larvae were observed. The emergence and hence survival rate of Limnephilus lunatus Curtis was significantly reduced by lindane at 0.1 ng l^–1, a value nearly five orders of magnitude lower than the 96-h LC₅₀. Parathion, with acute and subacute toxicity similar to that of lindane, did not significantly alter the emergence rate of this species. In contrast, this substance did produce a significant reduction in emergence rate of the closely related species Limnephilus bipunctatus Curtis at 1 ng l^–1, even though this species was significantly less susceptible than L. lunatus to parathion at high concentrations. We conclude that chronic insecticide exposure can be hazardous to freshwater macroinvertebrates even at unexpectedly low concentrations. The low-concentration effects may depend on both species and substance and therefore cannot be predicted from toxicity data at higher concentrations.     

Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C

Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [³H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.     

Structural and immunological reactivity of the principal neutralizing determinant V3 of glycoprotein gp 120 of HIV-1

The variable domain V3 in the outer glycoprotein gp 120 of HIV-1 is a highly important region with respect to immune response during the course of viral infection. Neutralizing antibodies are produced against this domain; in addition, it has been shown to be a functionally active epitope for T helper and cytotoxic T cells. The high degree of amino acid variability in individual HIV-isolates, however, limits the use of the V3-domain in approaches to vaccine development. In order to characterize the residues important for antibody interaction and binding to MHC class I proteins, we constructed a consensus sequence of the V3-domain with broad reactivity [1] and used synthetic peptides derived from this consensus with individual residues altered to alanine. These peptides were used as antigens in ELISA tests to define the amino acids which are important for binding to human and rabbit/anti-peptide immunoglobulins. In addition, we used these alanine-derived peptides in interaction studies with human HLA-A2.1 and mouse H-2D^d by testing their capacity to stabilize the respective MHC class I protein complexes on the surface of mutant cell lines T2 and RMA-S transfected with D^d gene. The experimental tests allowed us to define individual residues involved in antibody and MHC-protein interaction, respectively. In a further approach, we used those results to design interaction models with HLA-A2.1 and H-2D^d. Therefore, a structural model for H-2D^d was built that exhibits an overall similar conformation to the parental crystal structure of HLA-A2.1. The resulting interaction models show V3-peptide bound in an extended β-conformation with a bulge in its centre for both H-2D^d and HLA-A2.1 complexes. The N- and C-termini of V3 peptide reside in conserved pockets within both MHC-proteins. Anchoring residues could be determined that are crucial for the binding of the respective MHC class I haplotype. The cross-reactivity of V3-peptide in enhancing the expression of two different MHC class I molecules (H-2D^d and HLA-A2.1) is shown to be based on similar peptide binding that induces an almost identical peptide conformation.     

Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor

Cells of the obligately lithotrophic species Nitrosomonas europaea and Nitrosomonas eutropha were able to nitrify and denitrify at the same time when grown under oxygen limitation. In addition to oxygen, nitrite was used as an electron acceptor. The simultaneous nitrification and denitrification resulted in significant formation of the gaseous N-compounds nitrous oxide and dinitrogen, causing significant nitrogen loss. In mixed cultures of N. europaea and various chemoorganotrophic bacteria, the nitrogen loss was strongly influenced by the partners growing under oxygen limitation. Under anoxic conditions, pure cultures of N. eutropha were able to denitrify with molecular hydrogen as electron donor and nitrite as the only electron acceptor in a sulfide-reduced complex medium. The increase of cell numbers was directly coupled to nitrite reduction. Nitrous oxide and dinitrogen were the only detectable end products. In pure cultures of N. eutropha and mixed cultures of N. eutropha and Enterobacter aerogenes, ammonium and nitrite disappeared slowly at a molar ratio of about one when oxygen was absent. However, under these conditions cell growth was not measurable.     

The steady-state mRNA levels for thylakoid proteins exhibit coordinate diurnal regulation

Steady-state mRNA levels for thylakoid proteins were analysed in spinach cotyledons under diurnally changing light conditions. Most fluctuate considerably throughout the day, while the levels of others show only low amplitude or no oscillation. Levels of mRNAs coding for proteins that belong to the same multiprotein complex generally oscillate in parallel and exhibit maxima that are specific for that complex: mRNAs for photosystem I proteins appear prior to those for photosystem II polypeptides and these again prior to mRNAs for the three polypeptides constituting the oxygen-evolving complex. For the mRNAs that change with high amplitudes (e.g. those for LHCP or the 20 kDa apoprotein of the CP24 complex) oscillations have also been found under constant conditions, indicating that a circadian oscillator is involved. Transgenic tobacco seedlings harbouring chimeric GUS gene fusions with 5-flanking sequences from the spinach genes Lhcb, PsaF and AtpD (encoding a light-harvesting chlorophyll a/b apoprotein of photosystem II, subunit 3 of photosystem I and subunit of the plastid ATP synthase, respectively) confirm that the differences in the amplitudes as well as the timepoints of maximum mRNA accumulation are perceived via cis-regulatory elements upstream of the respective ATG codons.     

A Novel Class of RanGTP Binding Proteins

The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran''s GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.The nuclear pore complexes (NPC)¹ are the sites where the exchange of macromolecules between nucleus and cytoplasm occurs (Feldherr et al., 1984). Transport through the NPCs is bidirectional and comprises a multitude of substrates. Small molecules can passively diffuse through the NPC. The transport of proteins and RNAs >40–60 kD is, however, generally an active process, i.e., it is energy dependent (Newmeyer et al., 1986) and mediated by saturable transport receptors (Goldfarb et al., 1986; Michaud and Goldfarb, 1991; Jarmolowski et al., 1994). To accomplish multiple rounds of transport, these transport receptors are thought to shuttle between nucleus and cytoplasm (Goldfarb et al., 1986). An import receptor, for example, has to bind its import substrate initially in the cytoplasm, release it after NPC passage into the nucleus, and return to the cytoplasm without the cargo. Conversely, an export factor has to bind the export substrate only in the nucleus and on the way out. This model predicts asymmetry in these transport cycles and implies that the binding of the transport receptor to its cargo is regulated by the different environments of nucleus and cytoplasm.The nuclear import of proteins with a classical nuclear localization signal (NLS) is to date the best characterized nucleocytoplasmic transport pathway (for reviews see Görlich and Mattaj, 1996; Koepp and Silver, 1996; Schlenstedt, 1996). The signal contains one or more clusters of basic amino acids (for review see Dingwall and Laskey, 1991) and is recognized by the importin-α/β complex (for references see Sweet and Gerace, 1995; Panté and Aebi, 1996). The α subunit provides the NLS binding site (Imamoto et al., 1995; Weis et al., 1995) and is therefore also called the NLS receptor (Adam and Gerace, 1991). The β subunit accounts for the interaction with the NPC (Görlich et al., 1995; Moroianu et al., 1995) and carries importin-α with the NLS substrate into the nucleus. The translocation into the nucleus is terminated by the disassembly of the importin complex, and both subunits are returned probably separately to the cytoplasm. Importin-α interacts with -β via its importin-β binding domain (IBB domain; Görlich et al., 1996a ; Weis et al., 1996a ). Binding to importin-β with an IBB domain is sufficient for nuclear entry, and the IBB domain can therefore be regarded as the nuclear targeting signal of importin-α. The export domain of importin-α has not yet been identified, but it is distinct from the IBB domain.The small GTPase Ran (Drivas et al., 1990; Bischoff and Ponstingl, 1991b ; Belhumeur et al., 1993) plays a key role in NLS-dependent protein import (Melchior et al., 1993; Moore and Blobel, 1993). GTP hydrolysis by Ran is absolutely essential for import (Melchior et al., 1993; Moore and Blobel, 1993; Schlenstedt et al., 1995a ; Palacios et al., 1996) and is possibly even its sole source of energy (Weis et al., 1996b ). Although the molecular mechanism of import is far from being understood, it appears that Ran fulfils at least two distinct functions in this process: first, Ran''s GTP cycle probably drives translocation into the nucleus (Melchior et al., 1993; Moore and Blobel, 1993; Weis et al., 1996b ), which appears to involve the binding of (cytoplasmic) RanGDP to the NPC, followed by nucleotide exchange and GTP hydrolysis, but it does not involve binding of RanGTP to importin-β (Görlich et al., 1996b ). Unfortunately, nothing is known of how Ran''s GTP cycle would translate into a directed movement through the NPC. Secondly, Ran regulates the interaction between importin-α and -β (Rexach and Blobel, 1995; Chi et al., 1996; Görlich et al., 1996b ). Binding of RanGTP to importin-β disassembles the importin-α/β complex at the nuclear side of the NPC, thereby terminating translocation (Görlich et al., 1996b ). The asymmetric distribution of Ran''s principal GDP/GTP exchange factor (RCC1; Bischoff and Ponstingl, 1991a ) and GTPase activating protein (RanGAP1, or RNA1 in yeast; Bischoff et al., 1995a ; Becker et al., 1995) crucially determines where the importin heterodimer can form and where it is forced to dissociate. RCC1 is a nuclear, chromatin-bound protein (Ohtsubo et al., 1987, 1989) that generates RanGTP in the nucleus, whereas free RanGTP is depleted from the cytoplasm by RanGAP1, which is excluded from the nucleoplasm (Hopper et al., 1990; Matunis et al., 1996; Mahajan et. al, 1997). Thus, low RanGTP levels in the cytoplasm allow importin-α to bind -β, and the high RanGTP concentration in the nuclear compartment dissociates the importin complex. The concentration of free RanGTP can, in this model, be regarded as a marker for cytoplasmic identity (low RanGTP) and nuclear identity (high RanGTP), which is “sensed” by the Ran-binding site in importin-β.It is likely that at least some properties of importin-β are shared by the mediators of the other nucleocytoplasmic transport pathways. This is emphasized by the recent identification of the importin-β–related transportin (Pollard et al., 1996) as an import receptor recognizing the M9 domain, the nuclear targeting signal in hnRNP A1 (Michael et al., 1995), and of yeast transportin (Kap 104p) as an import receptor for mRNA binding proteins (Aitchison et al., 1996). Furthermore, importin-β or its NPC-binding domain cross-compete with other pathways, such as M9-dependent import, NES-mediated nuclear export, and the export of mRNA and U snRNA (Kutay et al., 1997). This would suggest that these other transport receptors share at least some binding sites at the NPC and take a similar path through the nuclear pore complex as importin-β.In addition to importin-β, a number of other Ran-binding proteins are detectable in eukaryotic cells, e.g., in overlay blots using Ran γ-[³²P]GTP as a probe. These can be grouped into two classes (Lounsbury et al., 1994, 1996): first, those with a RanBP1 homology domain including the Ran binding protein 1 (RanBP1) itself (Coutavas et al., 1993; Bischoff et al., 1995b ) and the nuclear pore protein RanBP2, which has four RanBP1 homology domains (Wu et al., 1995; Yokoyama et al., 1995). Their binding to Ran can be competed by RanBP1. Second, importin-β and so far unidentified protein(s) of ∼120 kD whose Ran-binding is competed by importin-β but not by excess of RanBP1 (Lounsbury et al., 1994, 1996). Both RanBP1 and importin-β inhibit the nucleotide exchange on RanGTP (Coutavas et al., 1993; Lounsbury et al., 1994, 1996; Bischoff et al., 1995b ; Görlich et al., 1996b ). However, they do not cross-compete with each other for Ran binding but instead bind to different, nonoverlapping sites on Ran (Chi et al., 1996; Kutay et al., 1997; Lounsbury and Macara, 1997). Another striking difference is that RanBP1 facilitates the activation of Ran''s GTPase by RanGAP1 (Beddow et al., 1995; Bischoff et al., 1995b ), whereas the importin-β/RanGTP complex is entirely GAP resistant (Floer and Blobel, 1996; Görlich et al., 1996b ).Although a direct involvement of Ran has so far only been demonstrated in the importin-dependent transport pathway, perturbations in the Ran system have severe effects on a great variety of cellular functions, such as RNA processing, RNA export, regulation of chromosome structure, cell cycle progression, initiation of replication, microtubule structure, etc. (for review see Dasso, 1993; Sazer, 1996). One could argue that these effects are all secondary consequences from an impaired NLS-dependent protein import. However, it is also possible that these defects are more direct and that eukaryotic cells contain many immediate targets of Ran function.Here we describe a novel superfamily of Ran-binding proteins, which includes about a dozen factors in yeast and probably even more in higher eukaryotes. The members of this superfamily share with importin-β an NH₂-terminal sequence motif that appears to account for RanGTP binding. Indeed we could confirm the interaction with Ran for the following factors: RanBP7 and RanBP8, two novel, related proteins described here, Cse1p, a cell cycle regulator in yeast, CAS, which is required for apoptosis in cultured human cells, and for Msn5p and Pse1p from yeast. Of these we have characterized RanBP7 and RanBP8 in more detail. Both resemble closely importin-β in their interaction with Ran, and both bind directly to nuclear pore complexes. RanBP7 can cross the nuclear membrane rapidly and in both directions. We provide evidence for a transport cycle in which RanBP7 first enters the nucleus, binds RanGTP inside the nucleus as a prerequisite for rapid re-export to the cytoplasm, after which the RanBP7/RanGTP complex becomes finally disassembled by the concerted action of RanBP1 and RanGAP1 in the cytoplasm. We propose that during these transport cycles, RanBP7 would normally carry an as yet unidentified cargo. This means, RanBP7 and possibly also the other members of the RanBP7/Cse1p/ importin-β superfamily could function as transport receptors that shuttle between nucleus and cytoplasm. RanBP7 and importin-β form an abundant, heterodimeric complex in the cytoplasm that appears to have a function different from nuclear import of proteins with a classical NLS. It might be a way to regulate either RanBP7 or importin-β function. Alternatively, the RanBP7/importin-β complex might constitute an import receptor with a substrate specificity different from that of the importin-α/β complex.     

A Conformation- and Phosphorylation-Dependent Antibody Recognizing the Paired Helical Filaments of Alzheimer's Disease

Abstract: Hyperphosphorylated tau (PHF-tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation-dependent epitopes on tau, and (c) those recognizing conformation-dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation-dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF-tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation-dependent phosphoepitopes on tau, typified by TG-3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site-directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG-3. Additional studies demonstrate that TG-3 reacts with neonatal mouse tau and PHF-tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG-3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.